Thioredoxin proteins are a superfamily of proteins that participate in redox reactions and are distributed among a wide range of living organisms (Holmgren, A. (1985) Ann. Rev. Biochem. 54:237-271; Eklund, H. et al. (1991) Proteins 11:13-28; Freedman, R. B. et al. (1994) Trends in Biochem. Sci. 19:331-336). The thioredoxin family active site is characterized by a CXXC motif (C represents cysteine and X represents any of the 20 amino acids incorporated into proteins). The neighboring cysteine residues cycle between a reduced sulfhydryl and an oxidized disulfide form.
The reduced form of thioredoxin activates some enzymes by reducing disulfide bridges that control their activity. In addition, thioredoxin is an electron donor in the reaction sequence that reduces ribonucleotides to deoxyribonucleotides catalyzed by ribonucleotide reductase (Stryer, L. (1995) Biochemistry 4th Edition, W. H. Freeman and Company, pages 677, and 750-751.). It has been reported that in humans, thioredoxin and the cellular redox state modified by thioredoxin play a crucial role in arterial neointima formation in atherosclerosis (Takagi, Y. et al. (1998) Laboratory Investigation 78:957-66). Thioredoxin is also thought to be involved in cellular defense mechanisms against oxidative damage (see, for example, Tanaka, T. et al. (1997) Laboratory Investigation 77:145-55). Thioredoxin is also thought to play a role in regulating glucocorticoid responsiveness by cellular oxidative stress response pathways by sensing the redox state of the cell and transmitting this information to the glucocorticoid receptor by targeting both the ligand- and DNA-binding domains of the receptor (Makino, Y. et al. (1996) Journal of Clinical Investigation 98:2469-77). Human thioredoxin has been suggested to be effective as a free radical scavenger and has been shown to limit the extent of ischaemia reperfusion injury (Fukuse, T. et al. (1995) Thorax 50:387-91).
Protein disulfide isomerases are an important class of thioredoxin family active site-containing proteins that catalyze the oxidation of thiols, reduction of disulfide bonds, and isomerization of disulfides, depending on the reaction conditions (Freedman, R. B. et al. (1994) Trends in Biochem. Sci. 19:331-336). Protein disulfide isomerases catalyze the formation of correct disulfide pairings in nascent proteins. Protein disulfide isomerases preferentially interact with peptides that contain cysteine residues but are otherwise undiscriminating. The broad substrate specificity of protein disulfide isomerases enables them to speed the folding of diverse disulfide-containing proteins. By shuffling disulfide bonds, protein disulfide isomerases enable proteins to quickly find the most thermodynamically stable pairings amongst those that are accessible. Consequently, protein disulfide isomerases are involved in protein processing, protein folding, and protein secretion.
The present invention is based, in part, on the discovery of a novel thioredoxin family member, referred to herein as xe2x80x9c22109xe2x80x9d. The nucleotide sequence of a cDNA encoding 22109 is shown in SEQ ID NO: 1, and the amino acid sequence of a 22109 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO:3.
Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes a 22109 protein or polypeptide, e.g., a biologically active portion of the 22109 protein, In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2. In other embodiments, the invention provides isolated 22109 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, wherein the nucleic acid encodes a fill length 22109 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs that include a 22109 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 22109 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 22109 nucleic acid molecules and polypeptides.
In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 22109-encoding nucleic acids.
In still another related aspect, isolated nucleic acid molecules that are antisense to a 22109 encoding nucleic acid molecule are provided.
In another aspect, the invention features, 22109 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 22109-mediated or -related disorders. In another embodiment, the invention provides 22109 polypeptides having a 22109 activity. Preferred polypeptides are 22109 proteins including at least one DnaJ domain and/or at least one thioredoxin domain, and, preferably, having a 22109 activity, e.g., a 22109 activity as described herein.
In other embodiments, the invention provides 22109 polypeptides, e.g., a 22109 polypeptide having the amino acid sequence shown in SEQ ID NO:2; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, wherein the nucleic acid encodes a full length 22109 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs which include a 22109 nucleic acid molecule described herein.
In a related aspect, the invention provides 22109 polypeptides or fragments operatively linked to non-22109 polypeptides to form fusion proteins.
In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 22109 polypeptides or fragments thereof, e.g., a DnaJ domain or a thioredoxin domain. In one embodiment, the antibodies or antigen-binding fragment thereof competitively inhibit the binding of a second antibody to a 22109 polypeptide or a fragment thereof, e.g., a DnaJ domain or a thioredoxin domain.
In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 22109 polypeptides or nucleic acids.
In still another aspect, the invention provides a process for modulating 22109 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 22109 polypeptides or nucleic acids, such as conditions involving inappropriate redox activity and/or aberrant protein folding.
The invention also provides assays for determining the activity of or the presence or absence of 22109 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.
In yet another aspect, the invention provides methods for modulating the redox activity or protein processing activity of a 22109-expressing cell, e.g., a hematopoietic cell. The method includes contacting the cell with a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 22109 polypeptide or nucleic acid. In a preferred embodiment, the contacting step is effective in vitro or ex vivo. In other embodiments, the contacting step is effected in vivo, e.g., in a subject (e.g., a mammal, e.g., a human), as part of a therapeutic or prophylactic protocol. In a preferred embodiment, the cell is a hyperproliferative cell, e.g., a hematopoietic cell.
In a preferred embodiment, the compound is an inhibitor of a 22109 polypeptide. Preferably, the inhibitor is chosen from a peptide, a phosphopeptide, a small organic molecule, a small inorganic molecule and an antibody (e.g., an antibody conjugated to a therapeutic moiety selected from a cytotoxin, a cytotoxic agent and a radioactive metal ion). In another preferred embodiment, the compound is an inhibitor of a 22109 nucleic acid, e.g., an antisense, a ribozyme, or a triple helix molecule.
In a preferred embodiment, the compound is administered in combination with a cytotoxic agent. Examples of cytotoxic agents include anti-microtubule agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an anti-metabolite, a mitotic inhibitor, an alkylating agent, an intercalating agent, an agent capable of interfering with a signal transduction pathway, an agent that promotes apoptosis or necrosis, and radiation.
In another aspect, the invention features methods for treating or preventing a disorder characterized by aberrant redox activity and/or aberrant protein folding in a 22109-expressing cell, in a subject. Preferably, the method includes administering to the subject (e.g., a mammal, e.g., a human) an effective amount of a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 22109 polypeptide or nucleic acid. In a preferred embodiment, the disorder is a cancerous or pre-cancerous condition.
In a further aspect, the invention provides methods for evaluating the efficacy of a treatment of a disorder, e.g., a cellular stress related disorder. The method includes: treating a subject, e.g., a patient or an animal, with a protocol under evaluation (e.g., treating a subject with one or more of: chemotherapy, radiation, and/or a compound identified using the methods described herein); and evaluating the expression of a 22109 nucleic acid or polypeptide before and after treatment. A change, e.g., a decrease or increase, in the level of a 22109 nucleic acid (e.g., mRNA) or polypeptide after treatment, relative to the level of expression before treatment, is indicative of the efficacy of the treatment of the disorder. The level of 22109 nucleic acid or polypeptide expression can be detected by any method described herein.
In a preferred embodiment, the evaluating step includes obtaining a sample (e.g., a tissue sample, e.g., a biopsy, or a fluid sample) from the subject, before and after treatment and comparing the level of expressing of a 22109 nucleic acid (e.g., mRNA) or polypeptide before and after treatment.
In another aspect, the invention provides methods for evaluating the efficacy of a therapeutic or prophylactic agent (e.g., an anti-neoplastic agent). The method includes: contacting a sample with an agent (e.g., a compound identified using the methods described herein, a cytotoxic agent) and, evaluating the expression of 22109 nucleic acid or polypeptide in the sample before and after the contacting step. A change, e.g., a decrease or increase, in the level of 22109 nucleic acid (e.g., mRNA) or polypeptide in the sample obtained after the contacting step, relative to the level of expression in the sample before the contacting step, is indicative of the efficacy of the agent. The level of 22109 nucleic acid or polypeptide expression can be detected by any method described herein. In a preferred embodiment, the sample includes cells obtained from a hematopoietic tissue.
In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 22109 polypeptide or nucleic acid molecule, including for disease diagnosis.
In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 22109 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 22109 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 22109 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.